Characterization of prostaglandin (PG)-binding sites expressed on human basophils. Evidence for a prostaglandin E1, I2, and a D2 receptor.

Recent data suggest that prostaglandins (PGs) are involved in the regulation of basophil activation. The aim of this study was to characterize the basophil PG-binding sites by means of radioreceptor assays using 3H-labeled PGs. Scatchard analysis for pure (greater than 95%) chronic myeloid leukemia (CML) basophils revealed two classes of PGE1-binding sites differing in their affinity for the natural ligand (Bmax1 = 217 +/- 65 fmol/10(8) cells; Kd1 = 0.5 +/- 0.2 nM; Bmax2 = 2462 +/- 381 fmol/10(8) cells; Kd2 = 47 +/- 20 nM; IC50 = PGE1 less than PGI2 less than PGD2 less than PGE2 less than PGF2 alpha) as well as two classes of PGI2 (iloprost)-binding sites (Bmax1 = 324 +/- 145 fmol/10(8) cells; Kd1 = 0.5 +/- 0.3 nM; Bmax2 = 2541 +/- 381; Kd2 = 27 +/- 6 nM; IC50 = PGI2 less than PGE1 less than PGD2 less than PGE2 less than PGF2 alpha. In addition, CML basophils exhibited a single class of PGD2-binding sites (Bmax = 378 +/- 98 fmol/10(8) cells; Kd = 13 +/- 4 nM; IC50: PGD2 less than PGI2 less than PGE1 less than PGE2 less than PGF2 alpha). In contrast, we were unable to detect specific saturable PGE2-binding sites. Primary and immortalized (KU812) CML basophils revealed an identical pattern of PG receptor expression. Basophils (KU812) expressed significantly (p less than 0.001) lower number of PGE1 (PGI2)-binding sites (Bmax1: 9% (20%) of control; Bmax2: 36% (50%) of control) when cultured with recombinant interleukin 3 (rhIL-3), a basophil-activating cytokine, whereas rhIL-2 had no effect on PG receptor expression. Functional significance of binding of PGs to basophils was provided by the demonstration of a dose-dependent increase in cellular cAMP upon agonist activation, with PGE1 (ED50 = 1.7 +/- 1.1 nM) and PGI2 (ED50 = 2.8 +/- 2.3 nM) being the most potent compounds. These findings suggest that human basophils express specific receptors for PGE1, PGI2 as well as for PGD2.     

Evaluation of prostaglandin-receptors in human and rat liver: interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50:2.5 +/- 1.7, (6.1 +/- 2.1) microM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9 +/- 0.9 (2.0 +/- 0.8) microM. The Scatchard analysis on [3H]PGE1- as well as on [3H]iloprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3 +/- 5.2 (21.3 +/- 4.3) fmol/mg protein and a Kd of 2.1 +/- 1.8 (1.9 +/- 0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4 +/- 18.2 (86.1 +/- 13.2) fmol/mg protein and a Kd of 10.5 +/- 2.9 (15.1 +/- 3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4 +/- 13.9 (35.9 +/- 8.2) fmol/mg protein and a Kd of 4.1 +/- 1.2 (1.7 +/- 1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3 +/- 42.1 (142.9 +/- 17.8) fmol/mg protein and a Kd of 16.3 +/- 4.9 (9.2 +/- 7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4 +/- 51.9 (38.8 +/- 7.4) fmol/mg protein and a Kd of 16.2 +/- 3.2 (2.5 +/- 1.2) nM. It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are able to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

Evaluation of prostaglandin-receptors in human and rat liver: Interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50: 2.5±1.7, (6.1±2.1) μM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9±0.9 (2.0±0.8) μM. The Scatchard analysis on [3H]PGE1- as well as on [3H]ilioprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3±5.2 (21.3±4.3) fmol/mg protein and a Kd of 2.1±1.8 (1.9±0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4±18.2 (86.1±13.2) fmol/mg protein and a Kd of 10.5±2.9 (15.3±3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4±13.9 (35.9±8.2) fmol/mg protein and a Kd of 4.1±1.2 (1.7±1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3±42.1 (142.9±17.8) fmol/mg protein and a Kd of 16.3±4.9 (9.2±7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4±51.9 (38.8±7.4) fmol/mg protein and a Kd of 16.2±3.2 (2.5±1.2) nM.It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are stable to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

3H]iloprost and prostaglandin E2 compete for the same receptor site on cardiac sarcolemmal membranes.

We have previously demonstrated that high-affinity PGE receptors are present on purified cardiac sarcolemmal (SL) membrane from bovine heart (Lopaschuk et al. (1989) Circ. Res. 65, 538-545). In this study we determined whether PGI2 receptors are also present on the cardiac SL membrane. Due to the extreme lability of prostacyclin (PGI2) under physiological conditions, the PGI2 analogue, Iloprost was substituted for PGI2. 3H-Iloprost specifically bound to two sites on the SL membrane; one of high affinity (Kd = 0.3 nM, Bmax = 97.0 fmol/mg SL), and one of lower affinity (Kd = 20.6 nM, Bmax = 1589 fmol/mg SL). Competition studies demonstrated that the concentrations of PGE2 and PGE1 necessary to displace 50% of the specific binding of 20 nM [3H]Iloprost on cardiac SL were 15-fold lower than the concentrations of unlabelled Iloprost necessary to displace 50% of binding. In contrast, a 15-fold higher concentration of unlabelled Iloprost was needed to displaced 50% of specific binding of 2 nM [3H]PGE2 compared to the concentrations of PGE1 or PGE2 required to displace 50% of [3H]PGE2 binding. In summary, our results indicate that a prostacyclin receptor is present on the cardiac sarcolemmal membrane, and that PGI2 competes for the same receptor site as PGE2.     

Characterization of gastric mucosal prostaglandin E2 receptor

1. The binding characteristics of gastric mucosal prostaglandin (PG) E2 (PGE2) receptor were investigated using mucosal cell membranes from rat stomach. The binding was found to be dependent upon PGE2 and membrane protein concentration, the time of incubation and the pH of the mixture, being highest at pH 3.0. 2. Scatchard analysis of the binding data revealed a curvilinear plot with high affinity binding (Kd = 2 nM; Bmax = 0.106 pmol/mg protein) and low affinity binding (Kd = 319 nM; Bmax = 2.262 pmol/mg protein) sites. 3. Competitive displacement study indicated that the receptor was specific for PGs of the E series, as PGF2 alpha and 6-keto-PGF1 alpha failed to displace the PGE2. 4. The study is the first report to provide biochemical parameters of specific PGE receptors in rat gastric mucosa.     

Increase in the number of atrial natriuretic hormone receptors in regenerating rat liver.

Forty-eight hours after partial (approximately 67%) hepatectomy the activity of the particulate guanylate cyclase was increased by 2-fold in the regenerating rat liver. This increase was not an artifact of membrane isolation procedures, and as determined by 125I-labeled Tyr-28 atrial natriuretic hormone-(1-28) ANF binding, was accompanied by a 2-fold increase in the number of ANF receptors. The Kd of the receptors in membranes of regenerating livers was not significantly different from the Kd of the receptors in livers of sham-operated rats. The linear synthetic descysteine analog of ANF, analog I, which binds only to the 66-kDa receptors, displaced approximately 40% of the specifically bound 125I-ANF in liver membranes from both hepatectomized and sham-operated (control) animals. Affinity cross-linking studies with 125I-ANF confirmed the increase in the 116-kDa ANF receptor in membranes of regenerating livers. In perfused livers derived from control and hepatectomized animals, the basal rates of cGMP production were not significantly different. However, atriopeptin II-stimulated cGMP production was twice as great in regenerating livers as compared with controls. These data demonstrate that the increase in particulate guanylate cyclase activity observed during liver regeneration is due to an increase in the 116-kDa ANF receptor-associated activity. Additionally, our data demonstrate that the regenerating rat liver may be a valuable model with which to study the role of the hepatic ANF receptor/particulate guanylate cyclase.     

Effect of topical application of estradiol-17 beta and PGE2 on PGE-binding sites in the porcine endometrium.

High- and low-affinity prostaglandin E2 (PGE2) binding sites were found on day 15 after estrus in the endometrium of cycling (Cy) and pregnant (Pr) gilts as well as gilts treated with intra-uterine Silastic beads containing estradiol-17 beta (E2) alone or in combination with PGE2 (E and PG gilts respectively) and inserted into the uterine lumen on day 10 of the cycle. The average apparent dissociation constants (Kd) and binding site concentrations (Bmax) for the high- and low-affinity sites were respectively (mean +/- SEM): 8.4 +/- 0.7 pM and 3.28 +/- 0.38 fmol/mg of protein and 5.3 +/- 0.8 nM and 71 +/- 9 fmol/mg of protein. Samples collected along the meso- and antimesometrial aspects did not differ (P greater than 0.05), although the low-affinity Bmax was higher on the antimesometrial aspect for Pr and Cy gilts only. No difference in Kd (P greater than 0.10) was found between treatments for high-affinity binding sites. For the low-affinity binding sites, Kd was higher for Pr compared to PG and E but not to Cy gilts (P less than 0.05). The high-affinity Bmax was higher (P less than 0.05) for PG, followed by E, Pr and Cy gilts (respectively: 5.50 +/- 0.26; 4.19 +/- 0.46; 1.78 +/- 0.40; 1.64 +/- 0.23 fmol/mg of protein), although Pr and Cy gilts were not different (P greater than 0.05). These results suggest that the localized presence of conceptuses in the uterus in early pregnancy does not markedly affect PGE binding sites but that intrauterine applications of Silastic beads containing E2 and PGE2 increase high-affinity Bmax and decrease low-affinity Kd.     

Modulation of 3H-prostaglandin E2 binding sites in the rabbit gastric mucosa

The binding of 3H-prostaglandin E2 (PGE2) to rabbit gastric mucosa was investigated. Binding depended on incubation time, temperature and pH, and was saturable and reversible. Scatchard plot analysis revealed a single class of binding sites with a dissociation constant (Kd) of 5.33 +/- 0.21 nM and a maximum number of binding sites (Bmax) of 138.1 +/- 3.4 fmol/mg protein. PGE1 and 16,16-dimethyl PGE2 potently competed with 3H-PGE2 for the binding sites of gastric mucosa, whereas PGA2, PGF2 alpha, 6-keto PGF1 alpha and thromboxane B2 were less potent. The gastric mucosa prepared from the rabbits given indomethacin (5 mg/kg s.c. three times) showed a lower Kd (2.47 +/- 0.19 nM) for 3H-PGE2 than that from untreated one. Treatment with a PGE1 analog, misoprostol (320 micrograms/kg s.c. three times) lowered the Bmax to 74.1 +/- 2.4 fmol/mg protein without any significant effect on the Kd value. It is concluded that rabbit gastric mucosa has specific binding sites for 3H-PGE2 which may be modulated by the levels of PGs in vivo.     

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.     

Adrenergic receptors in the liver parenchyma in children with chronic hepatitis]

In the present study adrenergic receptors have been investigated in liver parenchyma, obtained at the resection of extrahepatic portal hypertension children without parenchymal affection (control group, n-7) and the resection of children in parenchymal affection (group of chronic hepatitis children, n-6). It has been shown, that the binding of beta-adrenergic radioligand 3H-dihydroalprenolol (3H-DHA) in liver parenchyma membranes of both control and chronic hepatitis groups was saturable and showed high affinity. The Scatchard analysis of the binding data indicated that the binding site was characterized by Kd and Bmax of 1.2 +/- 0.5 nM, 261.2 +/- 50 fmol/mg, respectively, for the control group; and 0.9 +/- 0.15 nM, 68.5 +/- 18.8 fmol/mg, respectively, for the group of chronic hepatitis patients; (mean+SEM). The binding of alpha 1-adrenergic antagonist 3H-prazosin (3H-PRZ) in liver parenchyma was also saturable and showed high affinity. The binding site is characterized by Kd = 0.6 +/- 0.12 nM, Bmax = 92.8 +/- 8.0 fmol/mg, for the control group; and Kd = 0.8 +/- 0.15 nM, Bmax = 195.0 +/- 22.0 fmol/mg, for the group of chronic hepatitis. It has been found that the number of binding sites of 3H-DHA significantly decreased and the number of binding sites of 3H-PRZ did not change in chronic hepatitis liver parenchyma in comparison with the control group. The results obtained suggest the important role of beta-adrenergic receptors in the pathogenesis of chronic hepatitis and in liver regeneration in children.     

Stimulatory and protective effects of benzodiazepines on GABA receptors labeled with [3H]muscimol

We investigated the effects of benzodiazepines on [³H]muscimol binding to rat brain membranes and on heat inactivation of GABA receptors. Scatchard analysis of [³H]muscimol binding to frozen and 0.05% Triton X-100 treated membranes revealed two components; a higher affinity (Kd=2.2 nM, Bmax=1.2 pmol/mg protein) and a lower affinity component (Kd=15.9 nM, Bmax=4.4 pmol/mg protein). Diazepam and flurazepam (3 μM) increased significantly the specific binding of 40 nM but not of 2 nM [³H]muscimol. This stimulation was attributed to an increase in the affinity of the lower affinity component for GABA receptors. The time course of heat inactivation of GABA receptors revealed rapidly and then slowly denaturating Phases. These observations would suggest that there are multiple GABA receptors with different sensitivities to the heat treatment. Diazepam depressed remarkably the slowly denaturating phase(s). After heat treatment for 50 min, the single component of GABA receptors with Kd of 14.3 nM and Bmax of 0.6 pmol/mg protein survived, whereas in the membranes preincubated with 3 μM diazepam, the Kd and Bmax of the still viable GABA receptors were 14.8 nM and 1.14 pmol/mg protein, respectively. In light of these findings, the stimulation of the lower affinity component of GABA receptors may be related to the protective effect of these drugs against heat inactivation.     

Circadian rhythm in adenosine A1 receptor of mouse cerebral cortex

In order to investigate diurnal variation in adenosine A1 receptors binding parameters, Bmax and Kd values of specifically bound N6 - cyclohexyl-[3H]adenosine were determined in the cerebral cortex of mice that had been housed under controlled light-dark cycles for 4 weeks (light on from 7.00 to 19.00 h). Significant differences were found for Bmax values measured at 3-hr intervals across a 24-h period, with low Bmax values during the light period and high Bmax values during the dark period. The amplitude between 03.00 and 18.00 hr was 33%. No substantial rhythm was found in the Kd values. It is suggested that the changes in the density of A1 receptors could reflect a physiologically-relevant mechanism by which adenosine exerts its modulatory role in the central nervous system.     

Effect of piracetam administration on 3H-N-methylscopolamine binding in cerebral cortex of young and old rats.

Piracetam, a nootropic drug, has been used for some time in Alzheimer's disease for its facilitatory effect on learning and memory. Rats treated with piracetam (500 mg/kg, p.o.) daily, during 1 and 2 weeks, showed a significant increase in muscarinic receptor number (Bmax) and in the dissociation constant values (Kd) in the cerebral motor cortex, in binding studies using 3H-NMS as ligand. The effect was observed not only in young rats (control- Bmax = 663.4 fmol/mg protein, Kd = 0.45 nM; treated- Bmax = 961.9 fmol/mg protein, Kd = 0.82 nM) but also in aged animals (control- Bmax = 628.0 fmol/mg protein, Kd = 0.47 nM; treated-Bmax = 747.6 fmol/mg protein, Kd = 0.84 nM). Since piracetam does not interact with muscarinic receptors, the reason for its effect expressed as the enhanced number of brain muscarinic receptors is not clear but could be the result of stimulation of phospholipid synthesis and thus would represent an indirect action of the drug.     

Endothelial binding of transferrin in fractionated liver cell suspensions

Several studies using crude liver cell suspensions incubated with labeled transferrin have led to a conclusion that hepatocytes have transferrin receptors. When a visual probe, which permits evaluation of transferrin binding to individual cells, was used, the binding was unexpectedly found to be limited to endothelial cells in liver cell suspensions. Neither hepatocytes nor Kupffer cells contained transferrin receptors. In the present study, we fractionated liver cell suspensions using metrizamide gradients and centrifugal elutriation to obtain hepatocytes, Kupffer cell and endothelial cell fractions of high purity. Incubation of these fractions with 125I- or 59Fe-labeled transferrin led to exclusive binding to endothelial cells but not hepatocytes nor Kupffer cells. Kinetic analysis demonstrated Kd of 1.9 X 10(-7) M, Bmax of 3.1 pmol/10(6) cells per min, corresponding to 2.1 X 10(5) molecules/cell per min. At 4 degrees C, the binding reached a steady-state plateau within 5 min. Comparison of our data with those of previous investigators demonstrates a consistency if we consider that crude liver cell suspensions are contaminated with 2-3% endothelial cells. Thus, the previously reported findings may be entirely due to the contamination of crude liver cell suspensions with a small number of endothelial cells.     

Direct evidence for the presence of selective binding sites for (3H) prostaglandin E2 on rat peritoneal macrophages

A method is presented which provides for a simple and rapid determination of PGE2 receptors on viable peritoneal macrophages. Incubation of the harvested cells with (3H)PGE2 revealed specific binding of (3H)PGE2 by use of the Millipore filter assay system. Maximum binding was attained in the presence of 1 mM EDTA. Specific binding was saturable at 65 fmol/mg protein with an equilibrium dissociation constant (Kd) of 3.2 X 10(-8)M. Inhibition of (3H)PGE2 binding with unlabelled prostaglandins revealed a potency series of PGE2 greater than PGE2 greater than PGI2. The PGE2 concentration which displaced 50% of the labelled ligand was 10(-7)M. Comparable kinetic data were obtained for adenylate cyclase stimulation, since the concentration which showed a halfmaximal stimulation of cAMP production was 2 X 10(-7)M of PGE2. Since PGE1 and PGI2 compete with (3H)PGE2 binding in a non-parallel manner compared to PGE2 itself, it is proposed that macrophages possess different types of PG receptors.     

Retinal dopamine D1 and D2 receptors: Characterization by binding or pharmacological studies and physiological functions

1. In the retinal inner nuclear layer of the majority of species, a dopaminergic neuronal network has been visualized in either amacrine cells or the so-called interplexiform cells. 2. Binding studies of retinal dopamine receptors have revealed the existence of both D1- as well D2-subtypes. The D1-subtype was characterized by labeled SCH 23390 (Kd ranging from 0.175 to 1.6 nM and Bmax from 16 to 482 fmol/mg protein) and the D2-subtype by labelled spiroperidol (Kd ranging from 0.087 to 1.35 nM and Bmax from 12 to 1500 fmol/mg protein) and more selectively by iodosulpiride (Kd 0.6 nM and Bmax 82 fmol/mg protein) or methylspiperone (Kd 0.14 nM and Bmax 223 fmol/mg protein). 3. Retinal dopamine receptors have been also shown to be positively coupled with adenylate cyclase activity in most species, arguing for the existence of D1-subtype, whereas in some others (lower vertebrates and rats), a negative coupling (D2-subtype) has been also detected in peculiar pharmacological conditions implying various combinations of dopamine or a D2-agonist with a D1-antagonist or a D2-antagonist in the absence or presence of forskolin. 4. A subpopulation of autoreceptors of D2-subtype (probably not coupled to adenylate cyclase) also seems to be involved in the modulation of retinal dopamine synthesis and/or release. 5. Light/darkness conditions can affect the sensitivity of retinal dopamine D1 and/or D2-receptors, as studied in binding or pharmacological experiments (cAMP levels, dopamine synthesis, metabolism and release). 6. Visual function(s) of retinal dopamine receptors were connected with the regulation of electrical activity and communication (through gap junctions) between horizontal cells mediated by D1 and D2 receptor stimulation. Movements of photoreceptor cells and migration of melanin granules in retinal pigment epithelial cells as well as synthesis of melatonin in photoreceptors were on the other hand mediated by the stimulation of D2-receptors. 7. Other physiological functions of dopamine D1-receptors respectively in rabbit and in embryonic avian retina would imply the modulation of acetylcholine release and the inhibition of neuronal growth cones.     

Elevated level of beta-adrenergic receptors in hepatocytes from regenerating rat liver. Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.     

Analysis of ligand-binding data without knowledge of bound or free ligand molar concentration

A method is proposed to set the parameters in a nonlinear regression procedure to determine the equilibrium dissociation constant (Kd) and the high affinity receptor concentration (Bmax) of systems consisting of one ligand, one high affinity receptor, and n low affinity binding sites. This method is suitable when neither bound or free ligand formal concentrations nor the maximum of the binding signal can be deduced from the experimental data. The method makes use of (i) the abscissa of the first inflection point of the plot of any signal proportional to the binding of ligand to receptors versus the logarithm of the total ligand concentration, and (ii) the initial slope of the saturation curve plotted in direct coordinates. We first demonstrate that when such an inflection point exists, its abscissa lies between Bmax/2 + Kd(1 + d) and Bmax + 2Kd(1 + d), where d is a parameter representative of the binding to the low affinity sites. Second, we demonstrate that the initial slopes of two saturation curves in direct coordinates, where Bmax varies by a known factor, allows an estimation of the Bmax/Kd ratio, within certain limits. From these two sets of data it is subsequently possible to define a precise window for the values of both Bmax and Kd. The performance of the method has been evaluated in representative cases using Monte Carlo studies. The results establish conditions for the existence of an inflection point as well as the influence of low affinity binding, whether or not proportional to Bmax.     

Effects of N-phthalamoyl-L-glutaminic acid, a selective agonist of NMDA receptor, on binding of 3H-L-glutamate with synaptic membranes of the hippocampus]

In the present investigation the interaction of a novel selective NMDA receptors agonist, N-phthalamoyl-L-glutamic acid (PhGA), with the synaptic membranes preparation of human hippocampus was examined against NMDA. It was established that there are two binding sites of 3H-L-Glu, Kd1 = 0.35 +/- 0.11 nM, Bmax1 = 6.5 +/- 2.3 pmol/mg and Kd2 = 51 +/- 12 nM, Bmax2 = 98 +/- 17 pmol/mg. The inhibition constants (Ki) were calculated for NMDA and PhGA and were equal: Ki(NMDA) = 19 microM, Ki (PhGA) = 13 microM, respectively. It was concluded that PhGA is the partial agonist of the NMDA receptors.